Hrs Controls Sorting of the Epithelial Na⁺ Channel between Endosomal Degradation and Recycling Pathways^*

cDNA Constructs

Human α-, β-, and γENaC in pMT3 were cloned as described previously (30, 31). The following ENaC cDNAs were used: α-FLAG, β-FLAG, and γ-FLAG (each inserted at the C terminus) (6, 11); α_Y644A, β_Y620A, and γ_Y627A (9); α_Cl-1 (R177A, R178A) and α_Cl-2 (R175A, R177A, R178A, R181A, R190A, R192A, R201A, R204A) (16); and γ_Cl (R135A, R137A, R138A, R178W, R180A, K181A) and γ_Cl-G536C. Additional cDNAs used were human Nedd4-2 in pMT3 (12), Nedd4-2_WW (V210W, H212G, V367W, H369G, I440W, H442G, I492W, H494G) (6), Nedd4-2_C821A (6), Nedd4-2_S/T-A (S221A, T246A, S327A) (12), human SGK in pMT3 (12), mouse Hrs with C-terminal HA epitope in pcDNA3 and Hrs-ΔUIM (S270A, Q271A, S272A, E273A) (27), and ubiquitin-His (in pMT123) (6).

Cell Culture and Transfection

HEK 293T and COS-7 cells were cultured in Dulbecco's modified Eagle's medium. The cells were transfected with cDNAs encoding Hrs-HA, αβγENaC (one subunit containing a C-terminal FLAG epitope), and Nedd4-2 using Lipofectamine 2000 (Invitrogen). In some experiments, cells were cotransfected with SGK (see Fig. 3, C and D) or ubiquitin-His (see Figs. 4B and 7D). The total cDNA concentration was held constant using GFP cDNA (cDNA concentrations are reported in the legends for Figs. 1–8). Following transfection, 10 μm amiloride was added to the culture medium. In Fig. 3, A and B, cells were incubated in serum-free medium with or without dexamethasone (100 nm) for 4 h, and in Fig. 3, E and F, cells were treated with 200 μm 8-(4-chlorophenylthio)-cAMP sodium (Sigma), 100 μm 3-isobutyl-1-methylxanthine (Sigma), and 10 μm forskolin (Sigma) for 2 h prior to harvest. Cells were harvested 48 h after transfection.

Fischer rat thyroid (FRT) cells were cultured on permeable filter supports (Millicell PCF, 0.4-μm pore size, 12-mm diameter) in F-12 Coon's medium (Sigma) with 5% fetal calf serum (Sigma), 100 units/ml penicillin, and 100 μg/ml streptomycin at 37 °C, as described previously (32). 1 day after seeding, cells were cotransfected with αβγENaC (wild-type or mutant) and human Nedd4-2, with or without Hrs-ΔUIM (total cDNA was held constant with GFP cDNA) using TFX 50 (Promega). 1 h after transfection, 10 μm amiloride was added to the apical medium, and currents were recorded 48 h after transfection.

Coimmunoprecipitation

48 h after transfection, HEK 293T cells were lysed in 150 mm NaCl, 50 mm Tris (pH 7.4), 0.5% Nonidet P-40, and protease inhibitor mixture (Sigma). 500 μg of protein was immunoprecipitated with anti-FLAG M2 affinity gel (Sigma), anti-HA antibody (1:300, Sigma), or anti-WW1 antibody (1:50 (8)) with immobilized protein A (Pierce) or Ni-NTA beads (Qiagen). After extensive washing, the immunoprecipitated proteins were eluted in SDS-PAGE sample loading buffer (100 mm dithiothreitol, 20% glycerol, 100 mm Tris-Cl, pH 6.8, and 4% SDS) at 95 °C for 5 min and then separated by SDS-PAGE. Cell lysates (10 μg) were also separated by SDS-PAGE. Proteins were detected by immunoblot with anti-FLAG M2 monoclonal antibody-peroxidase conjugate (Sigma) at 1: 5000 dilution or anti-HA antibody (1:2000) and enhanced chemiluminescence (ECL Plus, GE Healthcare) and quantitated by densitometry using the ImageJ software.

Cell Surface Biotinylation

Transfected HEK 293T cells were washed with ice-cold PBS-CM (PBS with 1 mm CaCl₂ and MgCl₂) three times on ice, labeled with 0.5 mg/ml Sulfo-NHS-biotin (Pierce) in PBS-CM for 30 min on ice, and quenched by incubating with 100 mm glycine in PBS-CM for 10 min. After washing three times with PBS-CM, cells were lysed in 0.4% sodium deoxycholate, 1% Nonidet P-40, 63 mm EDTA, 50 mm Tris-HCl, pH 8, and protease inhibitor mixture. Biotin-labeled cell surface proteins were isolated by incubating cell lysate (200 μg) with immobilized NeutrAvidin beads (Pierce) for 12 h at 4 °C, detected by immunoblot (anti-FLAG M2 monoclonal antibody-peroxidase conjugate), and quantitated by densitometry using the ImageJ software.

To measure the rate of ENaC endocytosis, HEK 293T cells transfected with α_Cl-2-FLAG, β- and γENaC, Nedd4-2, and Hrs-ΔUIM or GFP were incubated with trypsin (5 μg/ml) for 5 min at 37 °C, as described previously (16). The cells were washed three times with PBS-CM to remove trypsin, incubated at 37 °C for times between 0 and 30 min to allow endocytosis of cleaved channels, and then placed on ice. Cleaved channels remaining at the cell surface were labeled with Sulfo-NHS-biotin, isolated with NeutrAvidin beads, detected by immunoblot, and quantitated by densitometry using ImageJ software.

To measure the rate of degradation of the cell surface fraction of ENaC, HEK 293T cells were biotinylated with Sulfo-NHS-biotin and then incubated at 37 °C for 0–120 min prior to lysis. Following lysis, remaining biotinylated ENaC was isolated with NeutrAvidin beads, detected by immunoblot, and quantitated by densitometry using ImageJ software (6).

Electrophysiology

Short circuit current was measured using modified Ussing chambers (Warner Instrument Corp.) in transfected FRT cells (12). The apical and basolateral surfaces were bathed in 135 mm NaCl, 1.2 mm CaCl₂, 1.2 mm MgCl₂, 2.4 mm K₂HPO₄, 0.6 mm KH₂PO₄, 10 mm HEPES (pH 7.4) at 37 °C. ENaC current was determined by subtracting current before and after the addition of 10 μm amiloride to the apical surface.

To quantitate ENaC endocytosis, FRT cells were transfected with α_Cl-1, β- and γ_Cl ENaC, and Hrs-ΔUIM or GFP, as described previously (16). Electrically silent ENaC channels at the cell surface were activated by proteolytic cleavage using trypsin (10 μg/μl), which was added to the apical membrane for 10 min and then removed by vigorous washes with ∼12 times the volume of the apical chamber. The time-dependent decline in ENaC current was measured every 5 min by the addition of amiloride (10 μm) to the apical membrane followed by washout of amiloride.

To quantitate ENaC recycling, FRT cells were transfected with α_Cl-1, β- and γ_Cl-G536C ENaC, and Hrs-ΔUIM or GFP. The apical membrane was incubated for 30 min with trypsin (10 μg/μl) at 37 °C to generate a pool of active ENaC that could undergo endocytosis. ENaC remaining at the cell surface was irreversibly blocked with 1 mm [2-(trimethylammonium)ethyl]methanethiosulfonate bromide (MTSET, Toronto Research Chemicals). After removal of MTSET, we monitored the rate of increase in benzamil-sensitive current (100 μm) as an assay of the return of active channels to the cell surface.

Statistics

Data are reported as mean ± S.E. Significance was determined by Student's t test (p < 0.05).

Interaction of ENaC with Hrs

To investigate the role of Hrs in targeting ENaC for degradation, we first tested whether Hrs binds to ENaC. In Fig. 1A, we cotransfected HEK 293 cells with α-FLAG, β- and γENaC and Hrs-HA. The immunoblots in the bottom two panels show that αENaC and Hrs proteins were expressed (the two αENaC bands correspond to the full-length and proteolytically cleaved forms). The top two panels show coimmunoprecipitation experiments. Following immunoprecipitation of αENaC, we detected Hrs by immunoblot only in cells in which ENaC and Hrs were coexpressed but not in cells expressing ENaC or Hrs alone (Fig. 1A, top panel). Likewise, when we immunoprecipitated Hrs, we detected a faint αENaC band (Fig. 1A, second panel). Together, the data indicate that Hrs interacts with αENaC.

Nedd4-2 catalyzes ENaC ubiquitination, which increases ENaC degradation in lysosomes (6, 18). We therefore tested the effect of Nedd4-2 on the ENaC-Hrs interaction. When cotransfected in HEK 293 cells, we found that Nedd4-2 increased binding of Hrs to αENaC but had no significant effect on total cellular αENaC or Hrs protein levels (Fig. 1A). Fig. 1B shows quantitation of αENaC that coprecipitated with Hrs and Hrs that coprecipitated with αENaC, as indicated. Mutation of the catalytic HECT domain (C821A) abolished the effect of Nedd4-2 (Fig. 1, A and B), indicating that ubiquitination is required for Nedd4-2 to modulate αENaC-Hrs binding. The effect of Nedd4-2 on binding was also abolished by mutation of its WW domains (Fig. 1, A and B), which mediate Nedd4-2 binding to ENaC.

In Fig. 1C, we asked whether Hrs would also bind to β- and γENaC. Similar to αENaC, we found that Hrs coprecipitated with both βENaC and γENaC. Moreover, Nedd4-2 increased Hrs binding to β- and γENaC (Fig. 1, C and D). Thus, Hrs interacts with all three ENaC subunits in a manner that is facilitated by Nedd4-2.

Liddle syndrome is caused by mutations that disrupt Nedd4-2 binding to ENaC PY motifs (9–11). In contrast to wild-type ENaC, Nedd4-2 failed to enhance the binding of βENaC to Hrs when the ENaC PY motifs were mutated (Fig. 2, A and B). Together with our similar finding when we mutated the Nedd4-2 WW domains (Fig. 1, A and B), the data suggest that the interaction between Nedd4-2 and ENaC is required for Nedd4-2 to modulate ENaC binding to Hrs.

Phosphorylation Regulates ENaC Binding to Hrs

ENaC trafficking is regulated by glucocorticoids and mineralocorticoids, in part through phosphorylation of Nedd4-2 (5). In Fig. 3, A and B, we tested the effect of the glucocorticoid dexamethasone on the interaction between ENaC and Hrs. Cells were cultured with or without dexamethasone for 4 h prior to lysis. In the absence of dexamethasone, Nedd4-2 increased αENaC binding to Hrs. In contrast, when cells were treated with dexamethasone, Nedd4-2 produced a smaller increase in binding (but had no effect on αENaC or Hrs protein levels).

Dexamethasone induces transcription of SGK (33, 34), which phosphorylates Nedd4-2 on three Ser/Thr residues, reducing its binding to ENaC (12–14). We therefore tested whether SGK would regulate the effect of Nedd4-2 on ENaC binding to Hrs. In cells transfected with SGK, Nedd4-2 failed to increase αENaC-Hrs binding, in contrast to cells not transfected with SGK (Fig. 3, C and D). This effect of SGK was dependent on its ability to phosphorylate Nedd4-2; mutation of the three SGK phosphorylation sites in Nedd4-2 (S/T) abolished the effect of SGK on ENaC-Hrs binding (Fig. 3, C and D). Together, the data indicate that SGK regulates the interaction between ENaC and Hrs through its phosphorylation of Nedd4-2.

Another Ser/Thr kinase, PKA, also phosphorylates Nedd4-2 and reduces its binding to ENaC (14). PKA is a downstream mediator of vasopressin, a hormone important in the regulation of epithelial Na⁺ and water transport (35). In Fig. 3, E and F, we modulated PKA activity by incubating cells for 2 h with or without cAMP agonists. In cells cotransfected with Nedd4-2, cAMP agonists reduced binding of αENaC to Hrs, similar to the effect of SGK. Thus, ENaC binding to Hrs can be modulated by at least two signaling pathways.

Interaction of Hrs with Nedd4-2

To further investigate the mechanisms by which Nedd4-2 regulates ENaC binding to Hrs, we asked whether Nedd4-2 and Hrs interact with one another. In Fig. 4A, we immunoprecipitated Nedd4-2 (anti-WW domain 1) and detected coprecipitated Hrs by immunoblot (anti-HA). We detected coprecipitated Hrs in cells cotransfected with Hrs and Nedd4-2. We also detected a small amount of coprecipitated Hrs in cells transfected with Hrs alone (Fig. 4A), likely through its binding to endogenous Nedd4-2. These findings support the conclusion that Nedd4-2 and Hrs form a complex.

Because Nedd4-2 is an E3 ubiquitin ligase, we tested the possibility that Nedd4-2 might catalyze Hrs ubiquitination. In Fig. 4B, we found that Nedd4-2 induced Hrs ubiquitination. However, ubiquitination did not induce Hrs degradation; in Figs. 1–3, Nedd4-2 failed to significantly reduce Hrs protein levels.

Effect of Hrs on ENaC Surface Expression

To test the role of Hrs in controlling ENaC surface expression, we overexpressed a dominant negative Hrs that lacks its ubiquitin-interacting motif (Hrs-ΔUIM). Previous work found that this construct disrupted the endosomal sorting of the epidermal growth factor receptor (36), similar to work with the yeast homologue Vps27p (23, 29). In Fig. 5A, we cotransfected α-FLAG and β- and γENaC in HEK 293 cells with or without Nedd4-2 and Hrs-ΔUIM and then detected the biotinylated cell surface fraction of αENaC by immunoblot. Nedd4-2 dramatically reduced αENaC surface expression but had minimal effect on αENaC in the total cellular pool, which primarily reflects ENaC in the biosynthetic pathway (6). Hrs-ΔUIM increased αENaC at the cell surface, reversing the effect of Nedd4-2 (Fig. 5A and quantitated in Fig. 5B).

To examine the functional consequence of this change in ENaC surface expression, we measured short circuit current in Fischer rat thyroid epithelia transfected with ENaC and Nedd4-2 with or without Hrs-ΔUIM. In the absence of Hrs-ΔUIM, Nedd4-2 inhibited amiloride-sensitive ENaC current in a dose-dependent manner (Fig. 5C). Hrs-ΔUIM reduced ENaC inhibition by Nedd4-2, shifting the dose-response relationship to the right. Thus, the functional and biochemical data indicate that the Hrs UIM is required for Nedd4-2 to regulate ENaC trafficking.

Effect of Hrs on ENaC Trafficking

Nedd4-2 regulates ENaC trafficking at two different steps in the endocytic pathway: endocytosis and lysosomal targeting (6, 16). To identify the step at which Hrs modulates Nedd4-2 function, we first asked whether Hrs-ΔUIM would alter ENaC endocytosis, using functional and biochemical approaches we previously described (16). In Fig. 6A, we used trypsin to acutely activate a furin-resistant (inactive) mutant ENaC expressed with Nedd4-2 in FRT epithelia. After removal of trypsin, we measured the rate of decay in amiloride-sensitive current as a measure of ENaC endocytosis. Current decay was rapid over the first 15–20 min and was identical in the presence or absence of Hrs-ΔUIM. As a biochemical correlate of this strategy, we measured the rate of disappearance of trypsin-cleaved furin-resistant channels from the cell surface using a biotinylation assay (Fig. 6B, and quantitated in Fig. 6C). Incubation of cells with trypsin for 5 min generated a pool of proteolytically cleaved α subunits, which were mostly removed over 15 min. Similar to the electrophysiological data, Hrs-ΔUIM did not alter the rate of disappearance of cleaved αENaC from the cell surface. Together, the data indicate that Hrs does not modulate ENaC endocytosis.

Once ENaC has reached the endosome, it can undergo degradation in lysosomes, or alternatively, recycle back to the cell surface to participate in Na⁺ uptake. To test whether Hrs plays a role in this sorting decision, we biotinylated ENaC at the cell surface and then measured the rate of degradation of biotinylated αENaC. When coexpressed with Nedd4-2, the majority of αENaC was degraded at 80 min, consistent with our previous work (6, 16). Hrs-ΔUIM abolished degradation of the cleaved (active) αENaC band (Fig. 7, A and B). This resulted in an accumulation of ubiquitinated ENaC (Fig. 7D).

In contrast to its effect on the cleaved form of αENaC, Hrs-ΔUIM did not prevent the disappearance of the full-length (inactive) form (Fig. 7C). This disappearance reflects two processes: degradation and proteolytic conversion to the cleaved form. We tested the possibility that Hrs selectively controls degradation of cleaved channels using the furin-resistant αENaC mutant that does not undergo cleavage. In Fig. 7, E and F, we biotinylated the cell surface fraction of channels and then detected biotinylated channels before or after an 80-min incubation at 37 °C. Nedd4-2 increased degradation of the uncleaved α subunit, but Hrs-ΔUIM did not prevent its degradation. Thus, Hrs functions in selectively targeting proteolytically cleaved ENaC to lysosomes for degradation.

By targeting ENaC for degradation, Hrs might alter the size of the ENaC pool available for recycling back to the cell surface. To test this possibility, we used an electrophysiological approach to quantitate the rate of ENaC recycling. Using a furin-resistant inactive ENaC mutant as a backbone, we introduced a cysteine into the channel pore (γ_G536C). In previous work, we found that covalent modification of this cysteine irreversibly blocks the channel (37). We activated channels at the cell surface with trypsin and then incubated the cells at 37 °C for 30 min to allow some of the channels to undergo endocytosis. We then irreversibly blocked channels remaining at the cell surface with MTSET and (following washout of MTSET and trypsin) quantitated the rate of current recovery as a measure of recycling of the trypsin-cleaved channels. We found that Hrs-ΔUIM increased ENaC recycling, as reflected by the enhanced rate of current recovery (Fig. 8).